Mechanistic Target of Rapamycin (mTOR) is a highly conserved serine/threonine protein kinase belonging to the PI3K-related kinase (PIKK) family (Wullschleger, et al. (2006). Cell 124, 471-484). mTOR forms two distinct kinase complexes, mTORC1 and mTORC2. mTORC1 controls cell growth and metabolism, in response to diverse cellular signals, including nutrients, growth factors and cytokines (Ma, X., and Blenis, J. (2009). Nat Rev Mol Cell Biol 10, 307-318). mTORC2 phosphorylates AKT at Ser473 and promotes cell survival (Sarbassov, et al. (2005). Science 307, 1098-1101). Recent advances in cancer genomic sequencing have revealed cancer mutations frequently target mTOR pathway, resulting in hyperactivation of mTOR signaling that drives uncontrolled cancer growth, metabolism and survival (Wood, et al. (2007). Science 318, 1108-1113). mTOR is an established molecular target for cancer therapy, because cancer cells tend to be addicted to aberrant mTOR signaling and mTOR inhibition is well tolerated (Bjornsti, M.-A., and Houghton, P. J. (2004). Nat Rev Cancer 4, 335-348; Guertin, D., and Sabatini, D. (2007). Cancer Cell 12, 9-22; Tsang, et al. (2007). Drug Discov Today 12, 112-124).
Since mTOR kinase inhibitors were described in 2008, numerous mTOR kinase targeting agents have been developed and entered into human clinical trials for cancer treatment (Zhang, et al. (2011a). Drug Discov Today 16, 325-331). The remarkable speed with which human clinical trials have been initiated and the sheer number of different compounds being tested in patients underscore the therapeutic potential of these inhibitors. Despite early promising results, major challenges remain. For example, a comprehensive, mechanistic understanding of these small molecule inhibitors is lacking. Additionally, while other small molecule kinase inhibitors have been proven clinically effective against malignancies in which kinase targets are hyper-activated, tumors typically develop drug resistance within six months after initial treatment. A major mechanism underpinning acquired resistance to kinase inhibitors is binding site mutations Gone, et al. (2001). Science (New York, N.Y.) 293, 876-880; Heinrich, et al. (2003). Journal of clinical oncology: official journal of the American Society of Clinical Oncology 21, 4342-4349; Kobayashi, et al. (2005). New England Journal of Medicine 352, 786-792). Thus, identification of resistant mutations is crucial for clinical diagnosis and development of new strategies to overcome resistant variants.
Thus, there is a need for additional tools to investigate mTOR kinase function, for the development and evaluation of mTOR kinase inhibitors and/or for the clinical diagnosis of drug-resistance. For example, there is a need for the identification of drug-resistant mutations in the mTOR kinase domain, which may be used as biomarkers for resistance and treatment decisions, as well as for the development and screening of new inhibitors.